双参宁心胶囊通过调控线粒体ATP敏感性钾通道减轻大鼠心肌缺血/再灌注损伤

Effect of Shuangshen Ningxin Capsule in Alleviating Myocardial Ischemia/Reperfusion Injury in Rats by Regulating Mitochondrial ATP-sensitive Potassium Channel

目的:观察双参宁心胶囊通过调控线粒体三磷酸腺苷(ATP)敏感性钾通道来减轻大鼠心肌缺血/再灌注损伤。方法:成年雄性SD大鼠56只,随机分为假手术组(sham),模型组(model),双参宁心组(SSNX)给予SSNX 90 mg·kg^-1,线粒体ATP敏感性钾通道(MitoKATP)通道抑制剂组(SSNX+5-HD),给予SSNX 90 mg·kg^-1和5-HD 5 mg·kg^-1;每组各14只。除假手术组外其余3组均阻断冠状动脉左前降支(LAD)45 min,分别于再灌注后3 h后处死,氯化2,3,5-三苯基四氮唑(TTC)染色观察心肌缺血和梗死面积,苏木素-伊红(HE)染色观察心肌组织损伤程度。试剂盒检测血清乳酸盐脱氢酶(LDH),肌酸激酶(CK)及肌酸激酶同工酶(CK-MB)的活性。透射电镜下观察心肌细胞线粒体及线粒体自噬超微结构变化,荧光探针法检测心肌细胞中线粒体膜电位水平的变化。结果:与sham组比较,model组心肌梗死面积与心肌缺血面积百分率明显增大,心肌组织排列紊乱,疏松,个别心肌纤维断裂,心肌细胞坏死,血清中CK,CK-MB,LDH活性显著升高(P<0.01),线粒体膜电位显著降低(P<0.01),透射电镜观察线粒体结构破坏明显。与model组比较,SSNX组心肌组织排列有序,少数区域细胞水肿轻度变性,心肌梗死面积与心肌缺血面积百分率显著降低,血清中CK,CK-MB,LDH活性显著降低(P<0.01),线粒体膜电位升高(P<0.01),SSNX+5-HD组心肌组织排列轻度无序,部分区域细胞水肿轻度变性,心肌梗死面积与心肌缺血面积百分率显著降低,血清中CK,CK-MB,LDH活性显著降低(P<0.01),线粒体膜电位升高(P<0.01)。而SSNX+5-HD组较SSNX组,血清中CK,CK-MB,LDH活性显著升高(P<0.01),心肌梗死面积与心肌缺血面积百分率显著增大,线粒体膜电位有所降低(P<0.05)。结论:双参宁心胶囊通过开放线粒体ATP敏感性钾通道来保护大鼠心肌缺血再灌注损伤。

Objective: To observe the effect of Shuangshen Ningxin capsule in alleviating myocardial ischemia/reperfusion injury in rats by regulating mitochondrial adenosine triphosphate(ATP)-sensitive potassium channels. Method: A total of 56 adult male Sprague-Dawley rats were randomly divided into sham-operated control group(sham),model group(model),Shuangshen Ningxin group(SSNX,90 mg·kg^-1). Shuangshen Ningxin and mitochondrial ATP-sensitive potassium channel(MitoKATP) channel inhibitor group 5-hydroxylacid group(SSNX+5-HD,5 mg·kg^-1),with 14 rats in each group. Except the sham operation group,the other three groups received occlusion of left anterior descending coronary artery(LAD) for 45 min,and were sacrificed 3 h after reperfusion. Myocardial ischemia and infarct size were observed by TSC Evans blue staining,and myocardial tissue damage degree was observed by hematoxylin-eosin(HE)staining. The kit was used to measure serum lactate dehydrogenase(LDH),creatine kinase(CK) and creatine kinase isoenzyme(CK-MB). The ultrastructural changes of mitochondria and mitochondrial autophagy were observed under transmission electron microscope. The changes of mitochondrial membrane potential in cardiomyocytes were detected by fluorescent probe. Result: Compared with the sham group,myocardial infarct size and myocardial ischemic area percentage in the model group were significantly increased,myocardial tissue arrangement was disordered and loose,individual myocardial fibers were broken,cardiomyocytes were necrotic,and serum CK,CK-MB, LDH activities were significantly increased(P<0. 01). Mitochondrial membrane potential was significantly decreased(P<0. 01), and mitochondrial structure was destroyed by transmission electron microscopy. Compared with the model group,the myocardial tissue of the SSNX group was arranged orderly,and a small amount of cell edema was mildly degenerated. The percentage of myocardial infarct size and myocardial ischemic area was significantly decreased, serum CK, CK-MB, and LDH activities were significantly decreased(P<0. 01),while mitochondrial membrane potential increased(P<0. 01). Compared with the model group,the SSNX+5-HD group had mild myocardial tissue disorder and mild degeneration of cell edema in some areas,the percentage of myocardial infarct size and myocardial ischemic area was significantly reduced,serum CK,CK-MB,and LDH activities were significantly decreased(P<0. 01),and mitochondrial membrane potential increased(P<0. 01). Compared with SSNX group,SSNX+5-HD group had significant increase in serum CK, CK-MB and LDH activities(P<0. 01), significant increase in the percentage of myocardial infarct size and myocardial ischemic area,and mitochondrial membrane potential Reduced(P<0. 05). Conclusion: SSNX protects rat myocardial ischemia-reperfusion injury by opening mitochondrial ATPsensitive potassium channel.